The human gene PIGA (phosphatidylinositol glycan class A) encodes a protein subunit of the a1-6-N-acetylglucosaminyltransferase complex, which catalyses an early step in the biosynthesis of the glycosyl phosphatidylinositol (GPI) anchor. In patients with Paroxysmal Nocturnal Hemoglobinuria (PNH), an acquired hemolytic anemia, a somatic mutation in the X linked PIGA gene occurs in a hematopoietic stem cell. Although PIGA -induced defects account for the deficiency of GPI-linked proteins on the cell surface of the affected blood cells, the basis for the expansion of defective hematopoietic progenitors in the bone marrow of PNH patients is unknown. Since PNH frequently evolves from Aplastic Anemia (AA) and since bone marrow failure is commonly associated with PNH, we hypothesize that the inability to link certain surface proteins to a GPI-molecule provides the clone with a growth advantage in a background of impaired hematopoiesis. The aim of the proposed research is to investigate this hypothesis and to study the growth behavior of PIGA negative cells in hematopoiesis. First we will therefore create an animal model in which the homologue murine Piga gene is inactivated in a proportion of hematopoietic progenitor cells just as in patients with PNH. To do this we will employ conditional gene inactivation using the Cre- loxP system of bacteriophage P1. A mouse that is able to inactivate its Piga gene in the presence of the Cre recombinase will be obtained by homologous recombination introducing two loxP sites into the Piga gene. Hematopoietic cells with a recombined, inactive Piga gene will occur in the progeny of matings between these mice and mice in which Cre expression is directed by a spatial and/or temporal specific promoter. As a result of Cre-dependent recombination of the loxP sites a proportion of cells will lack GPI-linked proteins. We will then compare PIGA(+) and PIGA(-) embryonic and adult hematopoiesis in the same mouse under a variety of circumstances, including the administration of selected cytokines along with agents known to injure bone marrow precursor cells. Competition between cells expressing wild type Piga and those expressing the recombined Piga allele will enable us to uncover even subtle differences in any stages of hematopoietic differentiation. In this way we hope to identify the factors that differentially influence the growth of PNH and normal hematopoietic progenitor cells and allow the PNH cells to become the dominate population while normal hematopoiesis is impaired. These experiments will increase our understanding of the functional role of GPI-linked proteins in hematopoiesis and provide new insights into the regulation of stem cell development in the bone marrow. The findings could provide the scientific foundation of new therapeutic modalities for PNH and other bone marrow failure syndromes.